Genetic Homology between Bacteria Isolated from Pulmonary Abscesses or Pyothorax and Bacteria from the Oral Cavity

ABSTRACT Pulmonary abscesses and pyothorax are bacterial infections believed to be caused primarily by oral microbes. However, past reports addressing such infections have not provided genetic evidence and lack accuracy, as they used samples that had passed through the oral cavity. The aim of this study was to determine whether genetically identical bacterial strains exist in both the oral microbiota and pus specimens that were obtained percutaneously from pulmonary abscesses and pyothorax, without oral contamination. First, bacteria isolated from pus were identified by 16S rRNA gene sequencing. It was then determined by quantitative PCR using bacterial-species-specific primers that DNA extracted from paired patient oral swab sample suspensions contained the same species. This demonstrated sufficient levels of bacterial DNA of the targeted species to use for further analysis in 8 of 31 strains. Therefore, the whole-genome sequences of these eight strains were subsequently determined and compared against an open database of the same species. Five strain-specific primers were synthesized for each of the eight strains. DNA extracted from the paired oral swab sample suspensions of the corresponding patients was PCR amplified using five strain-specific primers. The results provided strong evidence that certain pus-derived bacterial strains were of oral origin. Furthermore, this two-step identification process provides a novel method that will contribute to the study of certain pathogens of the microbiota. IMPORTANCE We present direct genetic evidence that some of the bacteria in pulmonary abscesses and pyothorax are derived from the oral flora. This is the first report describing the presence of genetically homologous strains both in pus from pulmonary abscesses and pyothorax and in swab samples from the mouth. We developed a new method incorporating quantitative PCR and next-generation sequencing and successfully prevented contamination of pus specimens with oral bacteria by percutaneous sample collection. The new genetic method would be useful for enabling investigations on other miscellaneous flora; for example, detection of pathogens from the intestinal flora at the strain level.

contains more than 700 bacterial species (5). Furthermore, bacteria in pulmonary abscesses are commonly collected via the respiratory tract using a bronchoscope, which makes it difficult to avoid contamination of the samples with oral bacteria (6). In addition, anaerobic bacteria may be affected by oxygen during bronchoscopic collection, resulting in a decrease in the recovery yield.
Many papers have discussed causative bacteria based on identification results from culture or a PCR-based method with specific primers; however, such identification does not provide a sufficient basis for causality. Demonstrating genetic homology is essential for clarifying causative bacteria. Owing to the recent advent and advancement of next-generation sequencing (NGS), which has made it possible to easily perform whole-genome sequencing for identification, we developed a novel method to confirm the presence of specific strains in the oral flora with strain-specific primers for PCR. The aim of this study was to determine whether genetically identical bacterial strains existed both in the oral microbiota and in pus specimens that were obtained percutaneously from pulmonary abscesses and pyothorax, without oral contamination.

RESULTS
Patient demographic information. Twenty-nine patients (19 with pulmonary abscess and 10 with pyothorax) were enrolled in the study between August 2013 and March 2015. A pus sample and an oral swab sample suspension were collected from each enrolled patient. The demographic information of the patients is shown in Table 1, and the workflow of the study is shown in Fig. 1. The patient cohort consisted of 24 males (15 with pulmonary abscesses and 9 with pyothorax) and 5 females (4 with pulmonary abscesses and 1 with pyothorax). The mean age was 64.6 6 13.8 years old. The main lifestyle factors included a history of smoking (76%) and high-risk alcohol consumption (34%). The main complications were diabetes mellitus (31%), chronic liver disease (17%), and malignancy (21%). The mean number of remaining teeth was 18.9 6 9.5, the mean oral hygiene index (OHI) was 2.2 6 2.4, the mean ratio of periodontal pockets $4 mm in depth was 18.6% 6 21.3%, and 21 patients had bleeding from the gingiva.
Identification of strains isolated from pus. A total of 49 bacterial strains were isolated from the pus samples of 29 patients. Bacterial isolates were identified by 16S rRNA gene sequencing ( Table 2).
Evaluation of genetic homology. A schematic of the workflow used to evaluate genetic homology in the current study is shown in Fig. 1. To determine whether the same bacterial strains were present in both the pus samples and oral swab sample suspensions of individual patients, we performed a two-step process that included the use of both "step 1 primer sets" and "step 2 primer sets." Among the 49 bacterial isolates (Table 2), we designed step 1 primer sets for 31 bacterial isolates that included 15 bacterial species (Table 3), and we excluded 11 isolates that could only be identified to the genus level, 3 isolates that failed in subculture, and 4 isolates that lacked genome Step 1 primers: based on the KEGG database information for the species isolated from pus, we selected or designed step 1 primer sets corresponding to each species isolated from pus. †, Step 2 primers: we designed five strain-specific primers for each strain based on the draft genomes of the strains isolated from pus and the genome information of related species listed in the KEGG database.
information of related species in the KEGG database or whose specific primers could not be designed ( Table 4). The homology rates of the selected 31 bacterial isolates were 99.2 to 100%. Evaluation of the samples by quantitative PCR (qPCR) using the step 1 primer sets showed that eight oral swab sample suspensions included more than 1 Â 10 4 copies/swab sample of the same species as were in the patient-matched pus isolates (Table 5). These bacterial species included Streptococcus intermedius (2 patients), Fusobacterium nucleatum, and Parvimonas micra from pulmonary abscesses and S. intermedius (2 patients), Streptococcus constellatus subsp. pharyngis, and Streptococcus constellatus subsp. constellatus from pyothorax. This indicated that identical strains may be present in both the pulmonary abscesses or pyothorax pus and the oral cavity microbiota. Next, we performed genome sequencing and de novo assembly of the eight strains isolated from pus. The genome sizes determined by de novo assembly of the draft genomes for the strains isolated from pus and their DDBJ accession numbers are shown in Table 6. We then designed five step 2 primer sets (strain-specific primers Sp1 to Sp5) for use in amplifying strain-specific sequences (approximately 2,000 bp/site). The step 2 primer sets were designed for each of the strains based on the draft genomes of the strains isolated from pus and genomic information. Total DNA extracted from the oral cavity swab sample suspensions of each of the eight patients was PCR amplified using the five step 2 primer sets for each strain (Table 7). Representative agarose gel electrophoresis results of the PCR amplicons are shown in Fig. 2. Amplified fragments of expected sizes were obtained for the total DNA from eight of the oral swab sample suspensions. The PCR amplicons from the oral microbiota samples were sequenced, and the results compared to the sequences of the pus isolates, as well as to those of the reference sequences from the GenBank, DDBJ, and EMBL databases. All PCR amplicon sequences of the oral microbiota samples completely matched (100% identity) the sequences from the strains isolated from pus. This confirmed that the sequences were identical between the strains isolated from pus and the oral amplicons, and there were no single-nucleotide polymorphism (SNPs), indels, or other variations identified. In contrast, there were no 100% matches for the sequences available in the open databases, for which homology ranged from 67% to 99.3% (Table 8).

DISCUSSION
In the current study, we demonstrated for the first time that genetically identical strains were present in both pulmonary abscess and pyothorax pus samples and swab samples of the oral microbiota. Furthermore, we were able to validate a new NGSbased approach for this type of study. We isolated 49 bacterial strains from the lesions of 29 patients by 16S rRNA gene sequencing, but we failed to determine the species for some isolates. Nevertheless, the 49 isolated bacterial strains included representatives of the reported causative microorganisms of pulmonary abscess or pyothorax,  such as Staphylococcus, Streptococcus, Parvimonas, Pseudomonas, and Fusobacterium (1-4). Among these isolates, four strains from pulmonary abscesses and four strains from pyothorax were subjected to our novel genetic homology analysis. PCR amplification of total DNA extracted from oral swab sample suspensions showed that the oral microbes shared 100% sequence homology with their patient-matched pus sample counterparts.
Anaerobes and microaerophilic streptococci have been suggested to be the causative organisms in most cases of pulmonary abscess or pyothorax, as the disease can be reproduced using anaerobes recovered from the gingival crevice and since multiple studies of transtracheal and transthoracic aspirates consistently implicate these bacteria (1,2). However, no report to date has shown genetic homology between clinical isolates from these types of lesions and the oral microbiota, which has largely been due to the lack of appropriate techniques. The "gold standard" method for determining genetic homology is pulsed-field gel electrophoresis, but this does not allow one-tomany DNA pattern comparisons between clinical isolates and the entire microbiota.
In this study, we developed a novel method for evaluating genetic homology. Whole-genome sequences of strains isolated from pus were created using an NGS method and then compared with those of the same or related species available in publicly accessible databases in order to develop strain-specific primer sets. To increase precision, five sets of strain-specific primers were synthesized for each strain isolated from pus. Total DNA extracted from oral swab sample suspensions was then amplified using these strain-specific primers, with all samples leading to the generation of PCR amplicons. This suggested that there were identical sequences in both the strains isolated from pus and the oral microbiota. To confirm this, each PCR amplicon was sequenced and the results used to match sequences from the oral microbiota to those  Table 4 for the primer sets. c The detection limit was $1 Â 10 4 copies/swab sample. d Anaerobic bacterium. of the strains isolated from pus. Our findings provide the first evidence of genetically identical bacteria being present in both pulmonary lesions and the oral cavity. Many bacterial species have been linked to pulmonary abscesses and pyothorax (1)(2)(3)(4). This is especially true for the Streptococcus anginosus group, consisting of S. intermedius, S. constellatus, and S. anginosus, which are common indigenous microbiota of the oral cavity and have all been shown to cause deep-seated organ abscesses (7-13). The S. anginosus group of microorganisms accounted for 35.5% (11/31 strains) of the bacterial isolates identified in the current study. Staphylococcus species were the next most frequently isolated bacterial strain (25.8%, 8/31 strains); however, we were unable to detect these bacterial isolates in the oral microbiota (Table 2). Thus, we suspect that these Staphylococcus isolates probably originated from the nasal cavity through the pharynx.
Bacteriological studies of pulmonary abscesses have always experienced significant challenges in sampling specimens without contamination from the oral cavity and oxygen exposure. Transtracheal aspiration and protected bronchial brushing are the most common procedures used to collect uncontaminated specimens (14). In the current a We designed 5 strain-specific primers (5 sites, Sp1 to Sp5, approximately 2,000 bp/patient) based on the draft genomes of the strains isolated from pus (14) and the genomic information of related species listed in the KEGG database. study, we sampled pus directly from the pulmonary abscess using percutaneous fineneedle aspiration (FNA) under X-ray fluoroscopic guidance, thereby completely avoiding the potential for contamination from the oral cavity. In addition, because our procedure was completed within one breath of the patient, it reduced the risk of air embolism and pneumothorax to the patient compared to the risk for computed tomography (CT) guidance and also minimized the effects of oxygen on the anaerobic bacteria.
We collected demographic information of the patients and performed oral examinations and found that the oral hygiene of the 29 patients in our study did not deteriorate: the mean ratio of periodontal pockets $4 mm in depth was 18.6% 6 21.3% and the mean OHI was 2.2. However, the mean number of decayed, missing, and filled (DMF) teeth (23 6 7.5) was higher than that of the general Japanese population (17.1 for the age range of 55 to 64 years and 19.2 for the age range of 65 to 74 years) and the mean number of residual teeth (18.9 6 9.5) was lower than the Japanese average (23.9 for the age range of 60 to 64 years and 21.6 for the age range of 65 to 69 years) (15). This indicates the relatively poor oral hygiene conditions of the patients and the possibility of complicating oral infectious sites, such as apical abscesses. The causative bacteria of purulent lung infections, such as F. nucleatum, P. micra, and S. anginosus, have been detected more often in apical abscesses than in saliva (16)(17)(18). In our study, we used oral suspensions to assess the oral microbiota, and we detected the species isolated from pus from a sufficient amount of bacterial DNA with the step 1 primers in only eight patients. If we had used samples from the possible infectious sites in the oral cavity, we might have been able to detect bacteria in a higher number of patients.  Table 7) were obtained for total DNA extracts in all samples (16 to 19 and 26 to 29). The sequences of the five amplicons were all 100% matched with the five specific sequences from the strains isolated from pus.
The current study has some limitations, in addition to the above-mentioned oral sampling method. The purpose of the study was to determine whether any causative bacteria of pulmonary abscesses or pyothorax were also present in the oral microbiota of the patient. That noted, we did not examine all possible causative bacteria. In other words, we did not consider bacteria that could not be cultured from the pus samples or were not listed in the KEGG database. Further studies would be helpful in creating a more robust data set.
In conclusion, our study provides the first demonstration that, in some cases, strains isolated from pus from a pulmonary abscess and pyothorax are genetic matches for DNA extracted from paired patient oral swab sample suspensions and, thus, are of oral origin. This finding provides strong evidence for the importance of oral care in preventing pulmonary abscesses and pyothorax. In addition, this new approach can be applied to other studies designed to detect pathogenic bacteria at the strain level in promiscuous microbiota associated with different diseases and is a robust tool for evaluating the underlying pathology of various infectious diseases.

MATERIALS AND METHODS
Study participants. This study was conducted after approval by the institutional review board of Himeji Medical Center on 21 June 2013. The enrolled participants included 29 patients with pulmonary abscess (n = 19) or pyothorax (n = 10) who visited the Himeji Medical Center respiratory medicine department between August 2013 and March 2015. Patients were assigned to the pulmonary abscess group when they presented pulmonary infections with a mass-like consolidation in the thoracic cavity or low-density areas within the lesions upon examination using contrast-enhanced chest CT. Those patients then underwent sampling of their lesions for isolation of bacteria. Patients were diagnosed with pyothorax when they exhibited symptoms of fever and pleural effusions, including bacterial contaminants.
Sample collection. For the collection of pus from pulmonary abscesses, the distance between the lesion and the skin was first measured using CT. Pus was collected by FNA using a 22-gauge needle inserted percutaneously under X-ray fluoroscopic guidance at the depth measured in advance on CT. The procedure was performed while the patient held a single breath for approximately 20 s. Pus samples from patients with pyothorax were collected from the thoracic cavity using a fine needle. Oral swab samples were obtained by scraping the whole oral cavity with an oral swab (Butler SG Sponge Brush; Sunstar, Inc., Osaka, Japan) wetted with sterile water.
Oral examination. Within 14 days of sampling, the number of residual teeth, the number of teeth with caries experience (decayed, missing, and filled [DMF] teeth), the oral hygiene index (OHI) (19), periodontal pocket depths (20), and bleeding on probing from subgingival pockets were evaluated by dentists.
Isolation of bacterial strains from pus samples. Each sample was smeared onto two plates of CDC anaerobe blood agar (Becton, Dickinson Japan, Tokyo, Japan). The plates were then incubated under aerobic or anaerobic conditions, and the resultant bacterial strains were isolated as pure cultures.
Identification of bacterial strains isolated from pus using 16S rRNA gene sequencing. Strains isolated from pus were identified by TechnoSuruga Laboratory (Surugaku, Shizuoka City, Shizuoka, Japan). The total length of 16S rRNA (approximately 1,500 bp) was amplified by PCR using the primer pair 9F/1406R, and then a sequence homology search was performed using partial sequences of approximately 500 bp on the 9F side to identify bacterial species. The homology search was performed using Apollo 2.0 DB-BA9.0 software (TechnoSuruga Laboratory) and sequences from the GenBank, DNA Data Bank of Japan (DDBJ), and European Molecular Biology Laboratory (EMBL) databases.
DNA extraction from oral swab samples. The tips of the oral swabs were aseptically cut from the swab shaft and mixed with 20 mL of sterile phosphate-buffered saline (PBS). After stirring this solution and removing the swab, it was centrifuged at 4°C and 13,000 rpm for 10 min to obtain pellets containing oral bacteria. The pellets were resuspended in 1 mL sterile PBS, and total DNA was extracted from 100 mL of the oral swab sample suspension using a Mora-extract DNA extraction kit (Kyokuto Pharmaceutical Industrial Co., Ltd.). The extracted DNA was resuspended in 100 mL Tris-EDTA (TE) buffer, and a final DNA solution at 10 ng/mL was prepared in TE buffer. qPCR using step 1 primer sets. As the initial step, we determined at the species level whether the same organisms were present among both the strains isolated from pus and the oral swab sample suspensions. Based on the information in the KEGG database regarding the species isolated from pus (21)(22)(23)(24), we selected or designed species-specific step 1 primers. The extracted DNA from the oral swab sample suspensions was evaluated using qPCR and the step 1 primers at TechnoSuruga Laboratory according to the method described by Takahashi et al. (25). The lower limit of detection of the PCR assay was 1 Â 10 4 copies/mL.
Acquisition of draft genomes of strains isolated from pus (genome sequencing and assembly). To determine the presence of the strains isolated from pus in the oral cavity at the strain level, we acquired draft genomes of the strains whose species were detected in the oral swab sample suspension using the above-described step 1 primer assay. Genomic DNA was extracted from the strains isolated from pus. After purifying DNA fragments from the agarose gel to remove contaminants, the genomic DNA was fragmented (550-bp insert) using the standard protocol 15041110 rev.C (December 2014) with the Illumina TruSeq nano DNA sample preparation kit (26), and then paired-end sequencing (2 Â 300 bp)